Rat anti cd11b clone m1 70

The following primary antibodies were used for immunostaining of optically cleared tissues: polyclonal rabbit anti‐α‐smooth muscle actin (SMA) (Abcam; ab5694; 1 : 300); and from (BioLegend UK Ltd, London, UK): rat anti‐CD45 clone 30‐F11 (103102; 1 : 300), CD11c clone N418 (117302; 1 : 200) and MHCII I‐A/I‐E clone M5/114.15.2 (107601; 1 : 300). The following macrophage markers were found to be unreliable with the tissue clearing protocols used: rat anti‐F4/80 (clone BM8), rat anti‐CD11b (clone M1/70), rat anti‐CD68 (clone FA‐11) (all from BioLegend) and rat anti‐F4/80 (clone Cl:A3‐1; from AbD Serotec, Kidlington, UK). The following secondary antibodies (all used at 1 : 500) were purchased from Invitrogen: goat anti‐rabbit Alexa Fluor 488 (A11008), goat anti‐rat Alexa Fluor 647 (A21247) and goat anti‐rat Cy3 (A10522); and from (Jackson ImmunoResearch, Ely, UK): goat anti‐Armenian hamster Cy3 (127‐165‐160).
For 2D analysis, SMA expression was detected using a mouse anti‐human SMA primary antibody (clone 1A4, Agilent; M0851) with a peroxidase‐conjugated ImmPRESS anti‐mouse IgG polymer detection kit (Vector Laboratories Ltd; MP‐7402, Peterborough, UK) using standard development with DAB. Mouse IgG1 was used for species‐ and isotype‐matched control (Agilent; X0931).

Conditional Hoxb8 immortalized neutrophil/macrophage committed myeloid progenitors, termed SCF-^condHoxb8 cells, were generated from bone marrow of WT, Bid^−/− and Ripk3^−/− mice as previously described [35 (link),45 (link),47 (link)]. Cells were cultivated in RPMI-1640 AQmedia complemented with 10% FCS, penicillin/streptomycin, 0.1 μM 4-OHT and 5% CHO/mmSCF-conditioned medium. Upon removal of 4-OHT, cells differentiated into mature neutrophils within 5 days. To confirm successful differentiation the following surface markers profile was used: Gr-1(Ly-6C/Ly-6G)^hi CD11b⁺ CD117(c-kit)^neg, using the following antibodies from BioLegend (San Diego, CA, USA): rat anti-CD11b (clone M1/70), rat anti-Gr-1 (clone RB6-8C5), and rat anti-CD117 (c-kit, clone 2B8). All experiments were performed with mature neutrophils.

Conditional Hoxb8 immortalized neutrophil/macrophage committed myeloid progenitors, termed SCF-^condHoxb8 cells, were generated from bone marrow of WT, Xiap^−/−, Ripk3^−/− and Xiap^−/−Ripk3^−/− mice as described previously.^{45 (link), 56 (link)} The cells were cultivated in RPMI-1640 AQmedia supplemented with 10% FCS, 1% penicillin/streptomycin, 5% SCF and 0.1 μM 4-OHT. Mature neutrophils were obtained within 5 days upon removal of 4-OHT from the medium. To confirm differentiation, neutrophils were stained for the surface marker profile Gr-1(Ly-6C/Ly-6G)^hiCD11b⁺CD117(c-kit)^neg, using the following antibodies (all from BioLegend, San Diego, CA, USA): rat anti-Gr-1 (clone RB6-8C5), rat anti-CD11b (clone M1/70) and rat anti-CD117 (c-kit, clone 2B8). All experiments were performed with mature neutrophils.